Untitled

Abstract

The immunosuppressive agent cyclosporin A (CsA) is known to cause cholestasis. Transcellular and paracellular transport of macromolecules contribute, albeit to a minor extent, to bile formation, but little is known about the effects of CsA on these pathways. The aims of this study were to investigate the influence of CsA on tight junction (paracellular) permeability and on transcytotic vesicular pathways labeled with horseradish peroxidase (HRP) in perfused rat liver. Livers from male Sprague-Dawley rats were perfused with Krebs-Henseleit buffer (albumin 1%, RBC 20%, and amino acid mixture). Taurodehydrocholate (1 microM/min) was coinfused into the portal vein; 1 microg/ml of CsA, dissolved in Cremophor-EL (CsA livers), or the vehicle alone (CEL livers), was added to the medium. Tight junction permeability was assessed by administering HRP (25 mg) as a short pulse to perfused rat livers, operating under single-pass conditions. Under such conditions, HRP output into the bile shows two components: an initial peak at approximately 3-5 min, corresponding to paracellular transfer across tight junctions, and a second peak at approximately 15 min, corresponding to vesicular transport. Furthermore, we assessed the vesicular transport pathway by examining HRP-labeled vesicles in the perisinusoidal (PS) and pericanalicular (PC) areas using ultrastructural morphometric analysis. To analyze HRP in hepatocytes and to study rapid and late transcytotic vesicular pathways, a 1-min pulse of a high dose of HRP (500 and 200 mg, respectively) was given. Two and 18 min after single-pass perfusion the livers were fixed with 2.5% glutaraldehyde-0.8% paraformaldehyde in 0.1 mM cacodylate buffer, pH 7.8. The total pericanalicular area, the HRP-containing structures, were quantified morphometrically in liver samples. At concentrations of 1.2 microg/ml, CsA produced a twofold increase in the paracellular transfer of HRP to bile. The areas under the second peak (transcellular vesicular pathway) of the biliary HRP secretion curve were similar in CEL- and CsA-treated livers. Morphometric analysis confirmed that CsA treatment did not affect the percentage area of HRP-labeled vesicles in either the pericanalicular or in the perisinusoidal area at 2 min (rapid pathway) and 18 min (late pathway). These results indicate that CsA increases tight junctional permeability whereas it does not inhibit rapid or late transcytotic vesicle pathways.

Receptor-Mediated and Fluid-Phase Transcytosis of Horseradish Peroxidase across Rat Hepatocytes

“…Although HRP appeared in bile roughly at the same time as FITC-dextran, the secretion rate revealed two peaks at 7 minutes and 15 minutes, respectively. These two peaks were seen irrespective of the HRP concentration perfused through the liver (0.08; 0.8; 8 mg/mL) and are in agreement with data from different laboratories [7, 8, 20, 21]. The bile flow, shown in Figure 2(b), remained constant at 1.6  μ L/g liver and min throughout this perfusion.…”

“…In contrast, we could demonstrate mannan inhibitable HRP secretion at 0.8 mg/mL. Thus, when HRP is applied at concentrations ranging between 1 mg/mL and 10 mg/mL as in morphological and perfusion studies [7, 8, 20, 21, 46] the proportion due to receptor-mediated endocytosis cannot be ignored, although at such HRP concentrations uptake by isolated hepatocytes is mainly by a fluid-phase mechanism.…”

Altered Expression of Tight Junction Proteins in Cyclosporine Nephrotoxicity

“…Thus, we suggest that pharmacologic doses of cyclosporine may induce an increase of occludin expression and a decrease in TER. Similarly, Lora et al [34] have demonstrated that, in the rat liver, low doses of cyclosporine significantly increase the paracellular permeability across the TJ. Cyclosporine-induced increases in TJ permeability have also been demonstrated in rat hepatocyte couplets [35].…”

Silymarin as a New Hepatoprotective Agent in Experimental Cholestasis: New Possibilities for an Ancient Medication

“…It occurs in bile-duct ligation [142,152] and under conditions of oxidative stress induced by both menadione [148,153] and tert-butylhydroperoxide [154,155]. It is also impaired in several models of druginduced cholestasis, such as those induced by administration of E 2 17G [148,156], cyclosporin A [148,157], acetaminophen [158], cholephilic dyes [159], carmustine [160], alpha-naphthylisothiocyanate [161] and LC [127], or its cholestatic conjugate, TLC [148,162]. In human hepatopathies, substantial alterations of tight-junctional proteins occur in primary biliary cirrhosis (predominantly in bile ducts) and in primary sclerosing cholangitis (predominantly in hepatocytes) [163].…”